Several approaches aimed at the induction of donor specific tolerance in recipients of organ and tissue grafts have been explored in the past. Amongst these, the administration of donor-specific bone marrow (BM) (prior to or together with organ transplants) has yielded encouraging results in small animal experimental models. One of the major drawbacks of such strategy, especially when radiation conditioning of the recipient is used, is represented by the risk of development of graft-versus-host disease (GVHD), which is thought to be mediated by the activation of donor-derived, recipient-specific, cytotoxic T lymphocytes. The removal of T cells from the BM inoculum before transplantation is indeed characterized by reduced incidence of GVHD, but also by reduced rate of engraftment. Cell mediated cytolysis occurs via at least two pathways; one dependent on the release of a lytic protein,named perforin, from cytotoxic cells, the other dependent on contact of a surface protein, named Fas ligand, expressed on the surface of cytotoxic effectors, with the Fas protein (also called Fas receptor or APO-1) on the surface of target cells. It is the purpose of this proposal to evaluate the use of BM cells derived from donors that have impaired or deficient cell-mediated cytotoxic functions, in BM transplantation aimed at inducing donor specific tolerance to subsequent tissues and organ grafts. The availability of mice lacking either a functional perforin (perforin knock out) or a functional Fas pathway (gld/gld mice) will allow us to evaluate in detail the role of these cytotoxicity-mediating pathways in the establishment of engraftment, the induction of chimerism and donor specific tolerance in allogeneic and xenogeneic models of BM transplantation, as well as in the development of GVHD. We have encouraging preliminary data that suggest an important role of perforin in the induction of GVHD. Lethally irradiated allogeneic recipients, when transplanted with wild type BM, invariably develop lethal GVHD. When they receive BM from perforin knock out donors, full allogeneic reconstitution occurs, donor specific tolerance is achieved and no GVHD is observed. The addition of high T cell inocula from perforin KO donors, on the other hand, is accompanied by development of GVHD, although characterized by a delayed onset and a slower course. This implies a possible role for other pathways of cytotoxicity in GVHD development, and sets the rationale for the proposed studies that will also use Fas-ligand deficient donors, as well as double deficient (perforin and Fas ligand) donors. Specifically, we intend to examine: the role of perforin and Fas pathways in BM recipient reconstitution, chimerism establishment and donor-specific tolerance induction. Furthermore, we intend to evaluate the immune profile of successfully reconstituted allogeneic or xenogeneic chimeras. The knowledge gained on chimerism establishment tolerance induction and prevention of GVHD by utilizing cytotoxically deficient donors will help not only in understanding the basic mechanisms of these phenomena, but might also help in evaluating the potential use of similar protocols in large animals models, with the ultimate goal of transferring this approach to the clinical setting.